(1) Field of the Invention
The present invention relates to a method used as a preparatory step in the qualitative and quantitative analysis of liquid samples, and is particularly valuable when the substance to be determined is present in trace amounts. The invention also relates to a device for carrying out the method. By using the method and the device according to the invention, even small amounts (˜1 cell/ml) of microorganisms can be determined rapidly in liquid samples.
(2) Background of Invention
Most waterborne human pathogens cause infections and human disease via the ingestion of feces-contaminated water or food. Various human parasites and pathogens are transmitted via the human fecal contamination of water used for drinking, bathing, recreation, or washing/preparation of foods. To ensure good public health, there is a need for readily available methods to detect and enumerate trace pathogens in water rapidly (<1 hr).
While the presence of pathogens in water presents a significant public health concern, the recovery of pathogens from environmental samples is generally difficult. Many fecal pathogens are infective at densities so low that water sample collection and concentration is inconvenient. Also, unpredictable physiological and morphological changes are observed in these pathogens in response to nutrient limitations and environmental stressors, and these injuries cause the organisms to exhibit atypical reactions.
These organisms then require specialized handling for their resuscitation. In addition to pathogens, viable but uncultivable organisms may be present in the water sample.
The methods commonly used to detect these pathogens were initially designed for clinical, rather than environmental, samples. However, clinical isolates are usually provided an ideal environment in which needed nutrients as well as protection from harsh environmental conditions, such as cold, heat, damaging chemicals, and radiation, are readily supplied to the isolate. In contrast, environmental isolates are exposed to harsh environmental conditions and effectively compete with organisms naturally present and adapted to life in the environment.
Pathogenic organisms are rarely readily adaptable to prolonged survival in the environment. For this reason, fecal microbial water contamination is often assessed by testing for harder and more robust, but not necessarily pathogenic, microbes, referred to as indicator organisms, such as the coliforms, especially Escherichia coli, and Enterococcus species. Indicator organisms serve to indicate whether a given water supply may be generally contaminated with fecal material without actually testing for the presence of all enteric pathogens. This contamination is viewed as predictive of the potential presence of enteric pathogens (i.e., without the presence of fecal material, the chances of these indicator organisms being present are usually remote).
In water quality inspection, it is essential to detect both the types of bacteria in the water being tested and the number of bacteria included in a given unit quantity of the water. Detection of the number of bacteria in water is absolutely necessary for super-pure water, such as is required in manufacturing medicines, in which the number of bacteria must be limited to an extremely small value, or in the case of manufacturing semiconductor devices (especially super-LSI semiconductor devices), in which the number of bacteria per 10 cc of super-pure water is limited to ten or less.
In a conventional method of detecting the number of bacteria in a unit quantity of super-pure water, a sample of the water is extracted and the bacteria in the sample are cultivated to allow them to multiply. The bacteria thus treated are then filtered and stained, and the stained bacteria are counted under a microscope.
When such a detecting method is employed, it takes about seven days to accomplish all of the steps, from sampling the super-pure water to counting the bacteria. If the quality of super-pure water is thereby determined unacceptable, semiconductor devices manufactured during the detection period must be discarded, which increases the average manufacturing cost of the devices.
The present inventors have proposed a bacteria counter that can detect the number of trace bacteria in water in an extremely short period of time, with the result that the manufacturing cost of semiconductor devices or the like can be reduced.
A need exists for a device or a system that solves the issues described above. The present invention fulfills those needs.
It is an objective of the invention to overcome or alleviate a problem of the prior art.